Báo cáo sinh học: " Identification and reciprocal introgression of a QTL affecting body mass in mice" pps

The estimated additive e ffect of the QTL on 10-week body mass was similar in both genetic backgrounds and in the original F 2population i.e.,∼0.4 phenotypic standard de-viations; no evid

INRA, EDP Sciences, 2004

DOI: 10.1051 /gse:2004018

Original article

Identification and reciprocal introgression

Julian K C a∗, Kellie A. R a,b, Sara A. K a, Pat M P a ,c, Fiona O a, Lutz B¨ a ,d

a Institute of Cell, Animal and Population Biology, University of Edinburgh,

Ashworth Laboratories, King’s Buildings, West Mains Road, Edinburgh, EH9 3JT, UK

(Received 12 January 2004; accepted 27 April 2004)

Abstract – The aim of this study was to examine the effects of a QTL in different genetic back-grounds A QTL affecting body mass on chromosome 6 was identified in an F 2 cross between two lines of mice that have been divergently selected for this trait The e ffect of the QTL on mass increased between 6 and 10 weeks of age and was not sex-specific Body composition analysis showed e ffects on fat-free dry body mass and fat mass To examine the effect of this QTL in di fferent genetic backgrounds, the high body mass sixth chromosome was introgressed

into the low body mass genetic background and vice versa by repeated marker-assisted

back-crossing After three generations of backcrossing, new F 2 populations were established within each of the introgression lines by crossing individuals that were heterozygous across the sixth chromosome The estimated additive e ffect of the QTL on 10-week body mass was similar in both genetic backgrounds and in the original F 2population (i.e.,∼0.4 phenotypic standard de-viations); no evidence of epistatic interaction with the genetic background was found The 95% confidence interval for the location of the QTL was refined to a region of approximately 7 cM

between D6Mit268 and D6Mit123.

quantitative trait loci / introgression / epistasis / body mass

1 INTRODUCTION

Many traits of medical, agricultural or evolutionary importance vary continuously rather than discreetly and numerous studies have identified

∗Corresponding author: julian.christians@ed.ac.uk

Present addresses:

b Aberdeen Centre for Energy Regulation and Obesity (ACERO), Energy Balance and Obesity Division, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen, AB21 9SB, UK

c Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK

d Animal Breeding & Genetics Dept., Animal Biology Division, Scottish Agricultural College, Bush Estate, Penicuik, EH26 0PH, UK

chromosomal regions that influence variation in a wide range of such traits

(i.e., quantitative trait loci, or QTL) [20] The vast majority of such studies

have examined QTL within a single genetic background, generally an F2 or backcross population between two lines or populations However, it would be expected that the effects of QTL would depend on the genetic background, given that the expressivity, penetrance and dominance of Mendelian mutations are frequently found to be affected by modifier genes [22] Although a num-ber of studies have found evidence of epistasis between QTL or marker loci

(e.g., [1, 5, 17, 24, 30]), the statistical power to detect epistasis is generally very

low [20] making it difficult to study specific interactions [24, 30] An alterna-tive to examining pair-wise interactions between loci in a mapping population

is to focus on a single QTL and to introgress the QTL alleles into different genetic backgrounds This approach is of greater relevance to marker-assisted introgression of beneficial alleles in agricultural species [12, 14, 31] and to the use of transgenic techniques to identify genes underlying QTL [10]

In the present study we have used the introgression approach to test for epis-tasis We have identified a QTL affecting body mass on chromosome 6 in an F2

cross between two lines that have been divergently selected for body weight Many studies have performed genome-wide scans for body mass QTL [7] and yet relatively few have gone on to replicate findings or to examine the effects

of QTL in different genetic backgrounds We therefore introgressed the sixth chromosome from each of the parental lines into the genetic background of

the other parental line, i.e., the ‘high’ QTL allele into the ‘low’ background and vice versa We tested the QTL in each of the parental lines because we

hypothesised that these might harbour modifier loci of the QTL For exam-ple, selection for high body mass would not only select for ‘high’ alleles at the QTL, but would also select for modifier alleles at other loci that either en-hanced the effect of the QTL’s ‘high’ allele, or reduced the effect of the ‘low’ allele

2 MATERIALS AND METHODS

2.1 Animal maintenance

Mice were fed a standard breeding diet (Rat and Mouse #3, Special Diet

Services, UK) ad libitum Further details regarding animal maintenance are

de-scribed in [16] The original F2population (described below) was raised in an older facility whereas in 1994 and 1995 all lines were transferred by embryo-transfer to a newly-built facility with a high health standard which resulted

in lower intergenerational variation in body mass [3] The introgression lines, the new F2 populations, and contemporaneous high and low lines (described below) were all raised in this new unit

2.2 Original F 2 population

The two parental lines consist of a high (PH) and a low body mass line (PL) derived from the same base population (an F1 derived from two inbred lines,

JU and CBA, crossed to an outbred line, CFLP) followed by divergent long-term (>50 generations) selection on protein mass and later on body weight at

70 days [3, 27] An F2population was derived from the inbred low line (gener-ation 7 post-inbreeding) and the outbred high line (gener(gener-ation 52 of selection; attempts to produce an inbred high line had been unsuccessful) [23] Body mass was recorded at 3, 6 and 10 weeks of age in 334 F2 individuals from first, second and third parity litters of 18 families To estimate fat content and fat-free dry body mass, animals were starved overnight and killed at 10 weeks

of age and then freeze-dried Carcass fat percentage was calculated using the following formula from [15] (see also [2, 4]):

Fat percentage =

[(freeze-dried weight× 1.13)/starved weight) − 0.302] × 100

2.3 Coarse genome-wide scan

Eighteen autosomal microsatellite markers (on chromosomes 3, 5, 6, 7, 9,

10, 12, 13, 14, 15, and 19) that were polymorphic between the high and low lines were identified and genotyped within the original F2population Markers

on other chromosomes were tested for polymorphism but the low frequency of polymorphism precluded a genome-wide scan

The association between genotype and body mass at 10 weeks of age was examined using single marker analyses Of the eighteen autosomal markers, the strongest linkage was with markers on chromosome 6 (data not shown), and therefore investigation of this chromosome was intensified and further poly-morphic markers were found (see below) to enable interval mapping More detailed examination of the X-chromosome is described elsewhere [19, 23]

2.4 Reciprocal introgression and new F 2 populations

To confirm the existence of the chromosome 6 QTL and to investigate its

effect in different genetic backgrounds, the QTL alleles from the high and low

Table I Genotyped microsatellite markers on Chromosome 6 Linkage map

posi-tions were calculated from the original and new F2 populations using CRIMAP [13] whereas physical map positions were obtained from the Ensembl Mouse Genome Server Database [8].

Marker name Genotyped in Linkage map Physical map

Original F 2 New F 2 position (cM) position (MB)

D6Mit216 Y Y 52.8 121.9

lines were introgressed into the opposite background by repeated backcross-ing, targeting the entire sixth chromosome To do so a new F1population was produced by crossing the contemporaneous inbred low (generation 28 post-inbreeding) and high lines (generation 24; an inbred high line was developed subsequent to the original F2 study) Reciprocal backcrosses were performed

to each of the parental lines (using both males and females as the recurrent parent) for three further generations, using only individuals that were

heterozy-gous at the following markers: D6Mit204, D6Mit159, D6Mit268, D6Mit123,

D6Mit261, D6Mit216, D6Mit111 and D6Mit15 These markers spanned most

of chromosome 6 with the largest gap being 25.6 cM between D6Mit261 and

D6Mit216 (see Tab I) To ensure that a double recombination had not occurred

within this gap, D6Mit105 was typed in the third backcross generation In this

way, the high allele was introgressed onto the low background to produce a

“high in low” (HinL) line and vice versa (LinH line) After three generations

of backcrossing (after the F1), the random contribution from the introgressed autosomal genome was expected to be about 6.3% (in addition to the intro-gressed sixth chromosome)

The third backcross generation was intercrossed (selecting only individuals

heterozygous at D6Mit105 and the eight markers described above) to produce

F2 populations within each of the lines In the LinH line, 10 families yielded

139 individuals from first, second and third litters, whereas in the HinL line the F2population consisted of 116 individuals from first through fifth litters of

6 families Body mass at 10 weeks of age was recorded at each generation and

in the new F2populations

2.5 Fixation of QTL allele in the introgressed lines

Preliminary results from the original and new F2populations indicated that

the QTL was proximal to D6Mit261 Therefore, to fix the QTL allele within

each of the introgression lines, individuals that were homozygous for the

intro-gressed allele at D6Mit204, D6Mit159, D6Mit268, D6Mit123 and D6Mit261

were mated with each other Six and four such matings were set up within the LinH and HinL lines, respectively, and offspring from these matings were weighed at 10 weeks of age

2.6 Microsatellite genotyping

Extraction of genomic DNA from either ear clip or spleen tissue and am-plification of microsatellite markers was performed by standard methods PCR products were separated on 20-cm polyacrylamide gels, stained with ethidium bromide, and photographed under UV light Photographs of gels were scored twice and ambiguous genotypes were re-amplified

2.7 Markers genotyped and linkage map

The chromosome 6 microsatellite markers genotyped are shown in Table I There are some differences between the original and new F2 populations be-cause not all of the markers were segregating in the original F2 population, and because more even spacing of markers was adopted for the new F2 popu-lation In the original F2population, only D6Mit204 and D6Mit17 were fixed

for different alleles in the two parental lines; as a result, some families are not

informative (i.e., not segregating) at the other markers All of the parents of the

new F2populations were heterozygous at all markers and thus all families are informative

The order of the markers was obtained from the Mouse Genome Database [21] and the Ensembl Mouse Genome Server Database [8]; this or-der was assumed to be correct since the genetic and physical maps are in agreement Map positions for these markers were calculated using the soft-ware package CRIMAP [13] combining genotypes from the original and new

F2populations (Tab I)

2.8 Interval mapping

Interval mapping (one- and two-QTL analysis) was performed using the

QTL Express package, which is able to accommodate outbred lines (e.g., the

high line in the original F2population) [26] These analyses fitted additive and dominance effects, as well as the effects of parental pair, sex and a linear co-variate for litter size at weaning simultaneously Significance thresholds for each trait were determined by permuting the marker data [6], using 1000 per-mutations Threshold F-values are presented as F0 05and F0 01for significance

atα = 0.05 and α = 0.01, respectively The two-QTL analysis estimates the effects of two QTL at separate positions simultaneously, examining all possi-ble pairs of locations (on a 1 cM grid), and determines the pair of locations for which the model explains the most variation; this analysis provides F-statistics

for the tests of two QTL versus no QTL and of two QTL versus the best

one-QTL model The significance of a second one-QTL was assessed by comparing the

two-QTL versus one-QTL model using the threshold for a single-QTL

analy-sis Confidence intervals for the location of QTL from interval mapping were calculated from 1000 bootstrapped samples [29]

2.9 Tests of epistasis

To test whether the effect of the QTL differed between the two genetic

back-grounds (i.e., high or low), a variation of the analysis described in [24] was

performed The additive and dominance coefficients at the position of the QTL (estimated by the combined analysis of the HinL and LinH F2 populations) were exported from QTL Express; the additive coefficient of an individual is the difference between the probabilities of being either homozygote, whereas the dominance coefficient is the probability of being a heterozygote Tests of epistasis were performed using a general linear model (GLM procedure, [25]) with 10-week body mass as the dependent variable and the additive and domi-nance coefficients, line (i.e., LinH or HinL) and the additive by line interaction

or the dominance by line interaction as the independent factors Parental pair (nested within line), sex and litter size at weaning were also included in these analyses

Scale is a potential problem in this type of analysis [9] We therefore tested for epistasis using the raw data as well as data standardised to have a mean of

0 and a standard deviation of 1 within each sex and population (see below)

Table II Means and standard deviations for body mass and composition at various

ages in the original and new F2populations.

Original F 2 population

LinH F 2 population

HinL F 2 population

3 RESULTS

3.1 Original F 2 population

The means and standard deviations for the measured growth traits are shown

in Table II The peak F value (13.6) from the interval mapping analysis of 10-week body mass exceeded F0 01(6.2); i.e., there was substantial support for

the presence of at least one QTL (Fig 1A), but the F-value for the addition

of a second QTL was not significant (F = 3.4 vs F0 05 = 4.6) As expected, homozygotes for the high-line allele had greater body mass than homozygotes for the low-line allele (Tab III); the dominance effect was not significantly

different from zero (Tab III) The 95% confidence interval for the location of the QTL determined by bootstrapping was 1 to 39 cM

When a sex by QTL interaction was included in the model, the estimated additive effects did not differ between the sexes (data not shown) When the sexes were analysed separately, the estimated locations did not differ signifi-cantly and fell within the 95% confidence interval obtained from the combined data (data not shown)

Interval mapping of 6-week body mass yielded similar results to 10-week body mass (Fig 1A; Tab III), although the estimated additive effect and sta-tistical support was lower (F = 9.0 vs F0 01 = 6.4) There was no significant linkage with 3-week mass (F = 3.2 vs F0 05 = 4.8) Fat-free dry body mass

Figure 1 A: F-value plot from interval mapping of 10-week (solid line), 6-week

(long-dashed line), 3-week (short-(long-dashed line), fat-free dry (dash-dotted line) and fat mass (dotted line) in the original F2population B: F-value plot from interval mapping of

10-week body mass in the LinH (solid line) and HinL (dashed line) F2 populations Both: Triangles denote the positions of markers typed The horizontal lines indicate the chromosome-wide 5% significance level obtained by permutation analysis.

at 10 weeks showed significant linkage (F= 12.8 vs F0 01 = 6.1) and the es-timated additive effect (in phenotypic standard deviation units) was similar to that for 10-week body mass (Tab III) While the estimated location of the QTL for fat-free dry body mass was substantially different than that for 10-week live body mass, the shape of the F-value plot was similar for both traits (Fig 1A) and the estimated location for the fat-free dry body mass QTL was within the 95% confidence interval for the 10-week live mass QTL Body fat also showed significant linkage (F = 6.6 vs F0 01 = 6.6), although the estimated

effect size in phenotypic standard deviation units was much smaller (Tab III)

new F 2 populations The additive e ffect is half the difference between the low-line and high-line homozygotes, where a positive value indicates that high-line > low-line The dominance effect is the difference between the heterozygote phenotype and the mean of the two homozygotes, where a positive value indicates that the heterozygote is closer to the high homozygote The e ffect in phenotypic standard deviation units is the absolute e ffect size divided by the standard deviation of the entire sample (using residuals from the sex-specific means so that the sexual dimorphism does not contribute to the standard deviation) The proportional e ffect size is the absolute effect multiplied by 100 and divided by the mean value for the trait Values are provided ± SE.

Original F 2 population

LinH F 2 population

HinL F 2 population

LinH and HinL combined

a For the analysis combining the LinH and HinL populations, the 10-week body mass data were first transformed to have a mean of 0 and a standard deviation of 1 within each sex and population; the resulting estimates are therefore in phenotypic standard deviation units.

The estimated location of the body fat QTL was just outside the 95% confi-dence interval for the 10-week live mass QTL and the shape of the F-value plot

differed substantially between body fat and the other traits However, given the estimated effect sizes and the sample size, the power to distinguish between linkage of two QTL affecting lean body mass and body fat separately versus

one pleiotropic QTL affecting both traits would be extremely low [18]

3.2 LinH and HinL line F 2 populations

In the original F2 population, the effects of the QTL were strongest for 10-week body mass (Tab III) and therefore in the new F2populations we focused exclusively on this trait In both the LinH and the HinL F2populations, body mass at 10 weeks showed a significant QTL (LinH: F = 7.7 vs F0 01 = 6.4; HinL: F= 6.6 vs F0 01= 6.2; Fig 1B) The absolute size of the additive effect was larger in the LinH line than in the HinL line (Tab III), but the average body mass in the former line was also much higher than in the latter (Tab II) When expressed as phenotypic standard deviation units, the estimated additive effects

in the two new F2populations are similar to that in the original F2population (Tab III) As in the original F2 population, the estimated dominance effects

in the new F2populations are not significantly different from zero (Tab III)

In two-QTL analyses, the F-value for the addition of a second QTL was not significant in either of the new populations (LinH: F = 1.6 vs F0 05 = 5.1; HinL: F = 1.5 vs F0 05 = 4.7) As in the original F2population, analysing the sexes separately yielded similar results (data not shown)

The estimated locations of the QTL in the two new F2 populations were very close, and about in the middle of the 95% confidence interval obtained for the original F2 population (see above) The 95% confidence interval for the location of the QTL determined by bootstrapping was 5 to 55 cM for the LinH line and 0 to 32 cM for the HinL line Because the estimated effects and locations of the QTL in the two new F2 populations were in such good agreement, the data were combined to obtain a better estimate of the location of the QTL Mean body mass and the absolute additive effect of the QTL differed between the two lines (Tabs II and III) and therefore the raw 10-week body mass data were transformed to have a mean of 0 and a standard deviation of 1

within each sex and population (i.e., by subtracting the mean and dividing by

the standard deviation of the appropriate sex and population) The estimated location of the QTL in the combined analysis was 16 cM and the F-value was highly significant (F = 14.1 vs F0 01 = 6.8); again there was no support for the existence of a second QTL (F = 2.7 vs F0 05 = 4.6) The 95% confidence